1. Field of the Invention
The present invention relates to a novel thermostable D-hydantoinase, and particularly to the nucleic acid sequence, amino acid sequence, vector constructs and the application thereof.
2. Description of the Related Arts
D-amino acids are usually called unnatural amino acids. They are not used as frequently as L-amino acids, but are becoming more and more important, especially in the field of pharmacy, e.g. in the preparation of penicillin, cephalosporin and xcex2-lactam antibiotic precursors.
Amoxicillin can be obtained by reacting D-p-Hydroxyphenylglycine (D-p-HPG) with 6-amino-penicillianic acid. Recently, the market for amoxicillin has expanded and grown more than 12% with each year. Due to the enhanced sales volume of the products, the demands for D-p-HPG precursors have increased markedly, with growth over 10% each year.
D-p-HPG was once chemically synthesized from D,L-p-hydroxyphenylhydantoin (D,L-p-HPH). However, this method has numerous disadvantages, including: (1) many reaction steps are required; (2) expensive solvents are employed; (3) products with low optical purity are obtained; (4) low yield is obtained in each reaction cycle; (5) complex steps are required for separation of racemizing residues; and (6) environmental contamination is induced during chemical processes (Takahashi et al., 1979, J. Ferment. Tech. 57:328-332). Yamada et al. (1978, J. Ferment. Tech. 56:484-491) developed a new process for the production of D-amino acids by unsymmetrical transformation of D,L-5xe2x80x2-substituted hydantoin with the D-hydantoinase in microorganisms, followed by treatment with nitrous acid. The products obtained by such chemo-enzymatic process are more economical and convenient. In addition, no measure to ensure optical purity are taken during this process.
Although the chemo-enzymatic process solves the problem in purity in the optical compound, many steps are still required, thereby increasing production costs. In addition, the reaction temperature has to be brought to below 20xc2x0 C. when the step of chemical decarbamoylation is carried out, otherwise D-p-HPG will be further hydrolyzed into 4-hydrobenzaldehyde. For a more convenient production method, many studies have become involved in searching for a microorganism capable of directly hydrolyzing substrates into D-amino acids. Olivieri et al. (1979, Enzyme Microb. Tech. 1:201-204) found that the crude extracts of Agrobacterium radiobater NRRL B 11291 possess the activity of N-carbamoyl-D-amino acid amidohydrolase, which can further hydrolyze N-carbamoyl-D-amino acid into D-amino acid. Therefore, the D-amino acids can be produced using D-hydantoinase and N-carbamoyl-D-amino acid amidohydrolase in microorganisms (Olivieri et al. 1981, Biotech. Bioeng. 23:2173-2183). Only one step is required in this method, and the racemizing residues can be completely converted to D-amino acids.
D-hydantoinase (EC 3.5.2.2) is useful in the production of N-carbamoyl-D-amino acids. Currently, microorganisms in which the D-hydantoinase gene has been cloned include Agrobacterium radiobater, Agrobacterium tumefaciens, Bacillus stearothermophilus, Bacillus thermoglucosidasius, Pseudomonas putida DSM 84, etc. The peptide length of D-hydantoinase ranges between 460 and 510 residues.
The two-step enzymatic process is desired for the production of D-amino acids in industries, thereby realizing cost savings for the manufacture. However, the current difficulty is the poor solubility of D,L-p-HPH. To overcome this problem, the reaction temperature has to be elevated to 50xc2x0 C. to increase the solubility. In the current cloned D-hydantoinases, however, there is a loss of or decrease in activity at this temperature. Only a few D-hydantoinases cloned from thermophilic Bacillus carry sufficient heat-resistance. See, for example, U.S. Pat. Nos. 5,523,224, 5,679,571, and 4,912,044. The microorganisms in which the heat-resistant D-hydantoinase has now been cloned include Bacillus stearothermophilus, and Bacillus thermoglucosidasius. However, some problems in stability and efficiency still exist in these enzymes when they are applied in the industrial process.
Therefore, there is still a need for a thermostable D-hydantoinase, in which the enzyme can maintain activities under high temperature (e.g. 50xc2x0 C.), and perform a preferred catalytic reaction, thereby converting 5xe2x80x2-substituted hydantoin to the desired D-amino acids.
The present invention provides a novel D-hydantoinase gene cloned from a thermophilic Bacillus circulans and an expression vector to largely express D-hydantoinase in a host. In addition, the present invention discloses the optimal conditions (i.e. divalent ions, pH, temperature) of enzyme activity for conversion of 5xe2x80x2-substituted hydantoin to the corresponding N-carbamoyl-D- and/or -L-xcex1/xcex2-D-amino acids.
It is therefore a primary object of the present invention to provide an isolated nucleic acid molecule. This molecule encodes for a protein consisting of the amino acid sequence set forth in SEQ ID NO:1, wherein the protein has D-hydantoinase activity and retains at least 50% activity after incubation at 50xc2x0 C. for 30 days.
Another object of the present invention is to provide a recombinant vector, which comprises the isolated nucleic acid molecule set forth above and a regulatory sequence.
Still another object of the present invention is to provide a thermostable D-hydantoinase, wherein the amino acid sequence of the D-hydantoinase is set forth in SEQ ID NO:1.
Yet another object of the present invention is to provide a method for preparing a D-amino acid, comprising reacting a 5xe2x80x2-substituted hydantoin with the thermostable D-hydantoinase of the present invention at a pH of 5 to 10 and at a temperature between 30xc2x0 C. and 90xc2x0 C.
Yet still another object of the present invention is to provide a method for the expression of a thermostable D-hydantoinase in a host cell, comprising (a) introducing the above recombinant vector into the host cell; and (b) culturing the transformed host cell under conditions sufficient to express the thermostable D-hydantoinase. The method can further comprise a step of purifying and recovering the resulting thermostable D-hydantoinase for the large production of thermostable D-hydantoinase of the present invention.